The present invention relates to methods of generating a functional mammalian single chain MHC class I complex in prokaryotic expression systems and a functional human single chain MHC class I complex in eukaryotic or prokaryotic expression systems, which complexes are capable of presenting specific antigenic peptides restricted to class I MHC and recognizable by specific CTL clones or CD+8 T-cells. The present invention further relates to a method of generating a functional mammalian single chain MHC class I-peptide complex in eukaryotic, or preferably prokaryotic expression systems, to nucleic acid constructs encoding said single chain MHC class I complexes and to a novel human single chain MHC class I polypeptide.
The major histocompatibility complex (MHC) is a complex of antigens encoded by a group of linked loci, which are collectively termed H-2 in the mouse and HLA in humans. The two principal classes of the MHC antigens, class I and class II, each comprise a set of cell surface glycoproteins which play a role in determining tissue type and transplant compatibility. In transplantation reactions, cytotoxic T-cells (CTLs) respond mainly against foreign class I glycoproteins, while helper T-cells respond mainly against foreign class II glycoproteins.
Major histocompatibility complex (MHC) class I molecules are expressed on the surface of nearly all cells. These molecules function in presenting peptides which are mainly derived from endogenously synthesized proteins to CD8+ T cells via an interaction with the αβ T-cell receptor [1-4]. The class I MHC molecule is a heterodimer composed of a 46-kDa heavy chain which is non-covalently associated with the 12-kDa light chain β-2 microglobulin. Class I MHC-restricted peptides, which are typically 8-10-amino acid-long, bind to the heavy chain α1-α2 groove via two or three anchor residues that interact with corresponding binding pockets in the MHC molecule. The β-2 microglobulin chain plays an important role in MHC class I intracellular transport, peptide binding, and conformational stability [5]. For most class I molecules, the formation of a heterodimer consisting of the MHC class I heavy chain, peptide (self or antigenic) and β-2 microglobulin is required for biosynthetic maturation and cell-surface expression [5].
Research studies performed on peptide binding to class I MHC molecules enable to define specific MHC motifs functional in displaying peptides derived from viral or tumor antigens that are potentially immunogenic and might elicit specific response from cytotoxic T lymphocytes (CTLs) [6,7].
The realization that CTLs have an important role in the control of many diseases, including chronic viral diseases, such as AIDS, and cancer have lead to an increased need to produce sufficient amounts of stable class I MHC complexes for functional and structural studies.
Soluble MHC molecules bound to various peptides are a valuable tool for the study of disease-related immune responses, for characterizing MHC-T-cell receptor (TCR) interactions [6], for structural studies [4], and more recently for direct visualization of antigen-specific T cells [8]. These molecules can be also used to activate specific CTLs in vitro as well as to study their phenotypic characteristics.
In recent years, various approaches have been used in attempts to develop an in vitro protocol for the induction of cytotoxic T cell responses against viral and tumor antigens [9-10]. To effectively activate T-cells, a high density of MHC-peptide complexes on the surface of the antigen presenting cells must be utilized [7-10]. Thus, a desirable approach for in-vitro T-cell activation would be to use soluble MHC-peptide complexes.
To overcome the low affinity binding of TCRs to soluble MHC molecules and as such to provide efficient T-cell activation, multimerization of the MHC-peptide complexes must be effected.
Soluble MHC multimers posses a higher avidity for T-cells since they provide multi-point binding of TCRs with their MHC-peptide ligands. As such, multimeric forms (tetramers) of MHC-peptide complexes have been the center of much interest recently, because they can be used for direct phenotypic characterization of T cell responses in normal as well as pathological conditions, thus, providing insight into the pathopysiology and mechanisms of various diseases.
However, such studies require a reproducible method for producing large amounts of soluble and functional multimeric MHC-peptide complexes. Thus, attempts were made to produce recombinant MHC class I and class II complexes [11-23] which are soluble and which can be produced in large quantities.
Early studies utilizing recombinant techniques, separately expressed the heavy chain and β-2 microglobulin components of the MHC complex in E. Coli and subsequently refolded them in-vitro in the presence of an antigenic peptide [11].
More recently, recombinant MHC complexes were expressed in eukaryotic expression systems and secreted therefrom in the form of a single polypeptide which included the heavy chain covalently linked to the β-2 microglobulin chain thus forming a stable and functional MHC complex which can be subsequently bound to a peptide of interest [12-15, 19, 21-23]. The expression of functional MHC complexes in eukaryotic cells suffers from several inherent limitations. Since the expressed polypeptides form a functional MHC complex they bind peptides endogenously derived from the cells utilized for expression and as such the purified MHC complex must be subjected to a peptide exchange step following purification [13]. In addition, the production yields of these single-chain MHC-peptide complexes was limited, typically reaching levels of several hundred micrograms per liter of culture supernatant.
The present invention provides a novel approach for the production of unprecedented large amounts of soluble, stable and functional MHC-peptide complex by utilizing high level bacterial expression of a single-chain MHC class I polypeptide or co-expression of the single chain MHC class I polypeptide and an MHC class I restricted antigenic peptide followed by in-vitro reconstitution of the scMHC class I-peptide complex via redox-shuffling and refolding in the presence of the antigenic peptide.
The present invention further provides a novel human single chain MHC class I polypeptide which is functional and which can therefore be utilized in either the monomeric or preferably the multimeric form to present MHC class I restricted antigenic peptides to CTL clones or to CD8+ T-cells from various sources.